Avidity, Potency, and Cross-Reactivity of Monoclonal Antibodies to Pneumococcal Capsular Polysaccharide Serotype 6B

ABSTRACT Many pneumococcal capsular polysaccharides (PSs) are similar in structure, and a pneumococcal antibody often binds to all of the PSs with a similar structure. Yet, these cross-reactive antibodies may bind to the structurally related pneumococcal capsular PSs with an avidity too low to be effective. If memory B cells producing such weakly cross-reactive antibodies are elicited with pneumococcal conjugate vaccines, the memory cells for low-avidity antibodies could compromise the subsequent immune responses to the cross-reactive PS (original antigenic sin). To investigate these issues, we produced 14 hybridomas secreting monoclonal antibodies (MAbs) to the capsular PS ofStreptococcus pneumoniae serotype 6B by immunizing BALB/c mice with antigens containing 6B PS and studied their epitope, avidity, in vitro opsonizing capacity, in vivo protective capacity, and “antigen binding titer” by enzyme-linked immunosorbent assay (ELISA) of 6A and 6B capsular PSs. Six MAbs bound to the non-cross-reactive 6B-specific epitope, and seven MAbs bound to the cross-reactive epitope present in both 6A and 6B PSs One MAb (Hyp6BM6) revealed a novel epitope. This epitope was found on 6A PS in solution, but not on 6A PS adsorbed onto the plastic surface of the ELISA plates. The avidity of the MAb for 6A or 6B PS ranged from 7.8 × 106 M−1 to 4.1 × 1011M−1. No MAbs were weakly cross-reactive, since none of the cross-reactive MAbs showed any tendency toward having less avidity to 6A PS (the cross-reactive PS) than to 6B PS. Avidity influenced the results of several antibody assays. When all of the hybridomas were examined, avidity strongly correlated with the titer of a unit amount of MAb to bind antigen-coated ELISA plates (r = 0.91) or to opsonize pneumococci in vitro (r = −0.85). Because both assay results are avidity dependent, the ELISA and the opsonization assay results were strongly correlated (r= 0.91), regardless of avidity. Avidity also correlated with the potency of a MAb to passively protect mice against pneumococcal infections. When only the immunoglobulin G hybridomas were examined, little increase in opsonizing capacity and in vivo protective potency was observed above 109 M−1. Taken together, an ELISA measuring antigen binding titer may be an adequate measure of the protective immunity induced with pneumococcal vaccines, and the absence of a partially cross-reactive MAb suggests that antigenic sin may not be significant in responses to vaccines against the S. pneumoniae 6B serotype.

The importance of the location of antibody binding on the M6 protein for opsonization and phagocytosis of group A M6 streptococci.

One of 19 mAbs against the native group A streptococcal M6 protein proved opsonic for type 6 organisms in a bactericidal assay. The opsonic and three nonopsonic antibodies were selected for isotype and complement fixation studies based on previous knowledge of their epitope site on the M6 molecule. While mAb 3B8 (IgG3), whose epitope is in the NH2-terminal hypervariable region of the molecule (distal from the cell), and mAbs 10B6 (IgG2a) and 10F5 (IgG2b), both located in the conserved central region of the molecule, all fix complement, 10A11 (IgG1) did not. Only mAb 3B8 was opsonic despite the fact that mAbs 10B6 and 10F5 both exhibited similar complement-fixing capacity, binding titer, and surface exposure of epitopes. Analysis of antibodies raised against synthetic peptides representing various regions of the M6 protein showed that only the amino-terminal peptide (residues 1-21) was capable of eliciting opsonic antibodies, despite the fact that peptides from other areas produced antibodies with high-binding titers to the native M6 protein and also with the ability to bind to intact streptococcal cells. These results not only support the observed type specificity of opsonic antibodies, but also clearly point to the importance of the location of antibody binding on the M molecule relative to the actual functional capacity of the antibody with respect to the opsonization and phagocytosis of M6 streptococci. These results may underscore the recently observed role of complement Factor H in the antiphagocytic activity of the M protein.